We have found that insulin can sequentially desensitize isolated rat adipocytes to the biologic affects of insulin by inducing both a loss of cell-surface insulin receptors and a post-receptor defect in the glucose transport system. To assess if insulin-induced receptor loss is a necessary step in the induction of the post-receptor defect, we propose experiments to determine if insuline can induce a post-receptor defect in the absence of receptor loss. Thus, we will correlate the extent of receptor loss with the magnitude of the post-receptor defect under various conditions to determine if a post-receptor defect can be induced by insulin without appreciable receptor loss. These results should further delineate the mechanism whereby insulin mediates insulin desensitization in adipocytes. By using a cultured rat adipocyte cell type for more chronic studies, we will examine the reappearance of "normal" insulin responsiveness after induction of a post-receptor defect in the transport system. These cultured cells will be characterized during development and differentiation with respect to insulin receptor number and affinity, insulins biologic effect to promote glucose transport, and the ability of cells to internalize insulin and undergo insulin receptor down-regulation. We have also found that when isolated adipocytes are incubated with insulin in medium rather than a simple buffer, no net loss of cell-surface insulin receptors is observed, although, insulin-receptor complexes are rapidly internalized and degraded through a receptor=mediated process. This finding suggests that insulin-receptors on adipocytes can recycle in vitro if they are incubated under more "physiological conditions. In this proposal we plan experiments to extend these observations in isolated adipocytes by: (1) characterizing the time and temperature kinetics of receptor recycling, and (2) determining the involvement of lysosomes and golgi in the recycling pathway. In addition, by performing more prolonged experiments with cultured rat adipocytes, we plan to test the hypothesis that insulin receptor recycling can chronically regulate the number of net cellular insulin receptors by affecting the equilibrium between the number of receptors internalized and the number recycled back to the plasma membrane.